Palm kernel meal regulates the expression of genes involved in the amino acid metabolism in the liver of Tibetan sheep

Background Palm kernel meal (PKM) is a by-product of oil palm kernel after oil extraction, which is widely used in animal feeds due to its high energy content. This study aimed to investigate the impact of supplementing Tibetan sheep with PKM on their hepatic phenotype, oxidative stress and immune response. A total of 120 Tibetan lambs (Initial weight = 12.37 ± 0.92 kg) were randomly assigned into four groups: control group (C group, 0% PKM diet), low group (L group, 15% PKM diet), middle group (M group, 18% PKM diet), and high group (H group, 21% PKM diet) on a dry matter basis. The feeding experiment was performed for 130 d, including a 10 d adaption period. Results Results showed that the level of GSH-Px were higher in the H and M groups than in the C and L groups (P < 0.05). The levels of IgM and TNF-α were higher in the M group when compared to those on the C group (P < 0.05). The level of IgA was significantly higher in the M group than in the H group (P < 0.05). Additionally, compared with the others groups, the hepatocytes in the M group displayed a radial arrangement, forming hepatic plates that were centered around the central vein. The transcriptome results revealed that proteasome 26 S subunit, ATPase 3 (PSMC3), proteasome 26 S subunit, ATPase 5 (PSMC5), proteasome 26 S subunit ubiquitin receptor, non-ATPase 4 (PSMD4), proteasome activator subunit 1 (PSME1), acyl-CoA dehydrogenase short/branched chain (ACADSB), enoyl-CoA hydratase, short chain 1 (ECHS1), serine dehydratase (SDS), ornithine transcarbamylase (OTC), and phenylalanine hydroxylase (PAH) were the hub genes regulating the amino acid metabolism in the liver. Conclusions In summary, dietary 18% PMK supplementation contributed to improve the hepatic phenotype, oxidative stress and immune response through regulating the expression of related genes.


Introduction
The rapid development of the livestock industry has caused an unprecedented increase in the demand for livestock resources [1].To alleviate the competition for food between humans and animals, it is necessary to explore alternative feedstuffs, with the development and utilization of agricultural by-product waste attracting increasing attention [2].By-products of agro-processing, a special form of agricultural resources, are widely used in various industries such as pig, laying hen, and cattle [3].
Among many agricultural by-products, palm kernel meal (PKM) has several advantages, including being abundant, cheap [4], rich in nutrition [5], abundant in vitamin E [6], and has antioxidation properties, among others.Thus, it has the potential to be used as an animal feed.Wyngaard [7] et al. found that adding 400 g/ kg PKC to the grazing dairy cows' concentrate had no effect on milk yield, fat, or protein content.Ribeiro [8] et al. showed that the addition of 19.5% palm kernel to lamb rations had no significant effect on the quality of lamb meat.At present, studies on palm kernel meal are all based on its digestibility, but animal utilization of key palm kernel meal nutrients is not well understood.
Based on studies concerning livestock, we hypothesized that including PMK in the diet might influence hepatic function in Tibetan sheep.Thus, the objective of this study was to investigate the impact of dietary PKM supplementation on hepatic phenotype, oxidative stress, and immune response in the livers of Tibetan sheep using RNA-Seq sequencing technology.

Animal diet and sample collection
One hundred and twenty healthy Tibetan lambs with an initial mean weight of 12.37 ± 0.92 kg (2 months old) were randomly selected from a commercial Tibetan sheep farm (Gonghe, Qinghai Province, China).According to dietary treatments, the lambs were randomly divided into four treatments, including a control group (C group, 0% PKM diet), low group (L group, 15% PKM diet), middle group (M group, 18% PKM diet), and high group (H group, 21% PKM diet) on a dry matter basis.The dietary concentrates of the four treatments contained 30% roughage (oat silage and oat hay to dry matter ratio of 1:1) and 70% concentrate.After a 10-day adaption phase, the Tibetan lambs were subjected to a 120-day formal trial having ad libitum access to food and water.The composition of the basic diet and nutrient levels is shown in Table 1.
At the end of the experiment, three Tibetan sheep in each treatment were randomly selected for slaughter in a commercial slaughterhouses.The liver tissue were collected and placed into liquid nitrogen for RNA extraction, while the remaining tissue samples were fixed in 4% paraformaldehyde for tissue-sectioning.

Antioxidant and immune response index assays
The immune response and antioxidant indices were measured using Enzyme-linked immunosorbent assay (ELISA).The immune response indicators included interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), immunoglobulin A (IgA), IgG, and IgM, while the antioxidant indicators included superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) levels.The ELISA kits used in this study were procured from Jiancheng Bioengineering Institute (Nanjing, China).

Histological analysis of the liver
The collected liver samples were dehydrated, trimmed, dipped in wax, embedded, sectioned, and then stained with hematoxylin-eosin (H&E).The randomly selected morphologically intact target areas of liver were observed by digital microscope (DP2-BSW, Olympus Corporation, Tokyo, Japan) at 400× magnification.The cross-sectional areas of the liver were determined using Image-Pro Plus software version 6.0 (Media Cybernetics Inc, Bethesda, MD, USA).

Analysis of gene expression
The expression of genes were estimated using string Tie software (http://ccb.jhu.edu/software/stringtie/,version: stringtie-1.3.4d) and ballgown (http://www.bioconductor.org).Here, the expression levels of all transcripts were estimated, and the expression abundance of mRNAs was performed by calculating fragment per kilobase of transcript per million mapped reads (FPKM) value.Principal component analysis (PCA) was performed using the princomp function in the R software (http://www.rproject.org/).Analysis for differential gene expression between different groups (and by edgeR between two samples) was performed using DESeq2 software.The threshold for differential gene expression was set at false discovery rate (FDR) ˂ 0.05 and absolute fold change ˃ 2.

Short time-series expression miner (STEM) analysis
The clustering of the differently expressed genes (DEGs) in the liver tissue of Tibetan sheep in different PKC groups was performed using the STEM software (version 1.3.11).Each gene was assigned to the closest profile using the Pearson correlation-based distance metric.
A permutation-based test was used to quantify the expected number of genes that would be assigned to each profile to determine the significance level of a given transcriptome profile [9].Profiles with a P value ˂ 0.05 were considered significantly enriched.Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactom Pathway Database were applied to identify the pathways regulated by the DEGs.In order to identify the core differential genes, the enriched genes were entered into the STRING database (version 9.1), and the Cystoscape plugin cytoHubba was used to detect the hub genes with the maximal clique centrality algorithm.

Antioxidation activities and immune response
The effects of the experimental treatments on antioxidation activities and immune response were showed in Table 2.The level of GSH-Px were higher in the H and M groups than in the C and L groups (P < 0.05).No significant difference in the levels of T-AOC and SOD among 4 groups were observed (P > 0.05).Additionally, the levels of IgM and TNF-α were higher in the M group when compared to those on the C group (P < 0.05).The level of IgA was significantly higher in the M group than in the H group (P < 0.05).

Histological observation
As illustrated in Fig. 1, varying supplement levels of PMK resulted in obvious changes in liver histomorphology.
Compared with the others groups, the hepatocytes in the M group displayed a radial arrangement, forming hepatic plates that were centered around the central vein.Additionally, these plates anastomosed with each other, forming a lost-like structure.

Transcriptome sequencing
The twelve complementary cDNA libraries produced a combined 4.94 million raw reads.After removing lowquality reads, 4.56 million high-quality clean reads were obtained from the twelve cDNA libraries.Sequences with Q30 ranged from 98.09 to 98.53%.The mapping rate for reads was ˃90%, with an average of 99.09% (Table 3), demonstrating the high quality of the reads and their suitability for further analyses.

Differentially expressed genes
We analyzed the gene expression of each sample based on the gene FPKM values.The results showed that the gene expression varied significantly among the groups, and the gene expression of the three parallel samples within the groups was always replicable (Fig. 2).PCA analysis of all the genes revealed that genes for samples in the same group clustered together, but samples for different groups clustered in different groups (Fig. 3).These results indicated that the expression of genes in samples  in the same groups was homogeneous but heterogeneous for samples in different groups.
After excluding the differentially expressed genes with expressions lower than 1000 FPKM, the remaining differentially expressed genes were analyzed by Upset.The results showed that the 3649 genes shared by the four experimental groups, with 49 genes being unique to group C, 874 genes being unique to group L, 56 genes being unique to group M, and 244 genes being unique to group H (Fig. 4).The results indicated that PKM supplementation in feeds dysregulated the expression of several genes.

STEM analysis
The expression profiles of 3649 DEGs were determined using cluster analysis based on STEM to obtain their expression patterns in the liver tissue of Tibetan sheep fed different PKM ratios.Forty-nine candidate genes were identified (Fig. 5), and the expression of eight of them was significantly dysregulated (P ˂ 0.05, Fig. 6).The eight gene profiles with significant enrichment were clustered into three groups.The first-group comprised four profiles: 40, 26, 38, and 37 (pink).The second-group consisted of two profiles, 27 and 29 (green), while 48 and 49 profiles were present in the third-group (blue).

KEGG analysis
The KEGG pathway enrichment analysis was performed based on the three groups clustered out by STEM analysis, and found that the enrichment to metabolic pathways in the first-group was mainly Glycolysis/Gluconeogenesis, Glycine, Serine, and Threonine metabolism, Cysteine, and Methionine metabolism ; the second-group was mainly enriched to Protein processing in the endoplasmic reticulum, Autophagy-animal, and Carbon metabolism pathway; and the third-group was mainly enriched to Ribosome, Oxidative Phosphorylation, and Endocytosis pathway (Fig. 7).The genes in the top three Pathways of the three groups were analyzed using the Reactom Pathway Database, and the results showed that the top Pathway was involved in the Metabolism of amino acids and their derivatives (Table 4).PPI analysis further revealed that PSMC3, PSMC5, PSMD4, PSME1, ACADSB, ECHS1, SDS, OTC, and PAH were the hub genes regulating the amino acid metabolism in the liver (Fig. 8).

Discussion
Palm kernel meal is rich in micronutrients such as vitamins E and B [10].Vitamin E is a fat-soluble vitamin that protects the liver from oxidative stress by reducing the production of free radicals and preventing damage to cell membranes [11].Our results showed that the liver antioxidant index GSH-Px was significantly higher in the 18% and 21% PKM diets than in the other two groups.Mariana et al. (2023) found that PKM presented bioaccessible compounds after digestion with antioxidant activity [12].Therefore, with increasing additions of PKM to the diet, an increase in the antioxidant bioaccessible compounds was also increased, thereby reducing the production of free radicals in the organism.Hepatocytes express innate immune receptors and present antigens to T cells to induce anti-inflammation.
PKM contains a mass of anti-nutritional factors, such as non-starch polysaccharides, trypsin inhibitors, etc.Studies have shown that a specific level of non-starch polysaccharides promoted the immune response, however, excessive supplement of PKM may result in the occurrence of disease [13].Our analysis showed that IgA and IgM were significantly higher in the 18% PKM diets, whereas reduced when fed the 21% PKM.Huang et al. (2024) showed that the replacement of wheat bran with PKM will promoted intestinal immunity [14].One possible explanation is that, dietary 21% PKM supplementation produced more accumulation of anti-nutrient, resulting in damage of immune system.Additionally, Our results showed that the TNF-α concentration was significantly higher in the 18% PKM supplement than in the other three groups.TNF is a proliferative factor secreted by the macrophages, which stimulated cyclic AMP (cAMP) generation, proliferation, and Ig production in B cells [15].Overall, we comprehensively analyzed that dietary 18% PKM supplementation exhibited a better oxygenation and immunization in the liver.
As a critical hub for numerous physiological processes, the liver participated in the nutrient metabolism, immune system and intestinal homeostasis [16].The functional structural unit of liver was hepatic lobule, the hepatic lobule will consist hepatocyte chords around the central vein.The hepatic chords are not tightly connected to each other, but appear as a net-like interstice, oxygen-rich blood from the hepatic artery mixes with nutrient-rich blood from the portal circulation in the sinusoid before flowing over the cells of the lobule and draining into the central vein [17].Therefore, the interstitial space of the hepatic chords can also indirectly reflect the nutrient exchange.In our results, the 18% PKM supplementation had a larger hepatic chord interstitial space and a more complex lost-like structure, which may contribute to the hepatic function.
Amino acid catabolism primarily occurs in the liver [18].RNA-Seq results showed that the DEGs in this study mainly participated in the metabolism of amino acids and their derivatives.Though PKM only contains 14-21% crude protein [19], the amino acid utilization of PKM is high [20].Except for valine and glycine, the digestibility of most amino acids is above 85% [21].The higher digestibility of palm kernel meal was probably the primary reason the differential genes were mainly enriched in the amino acids and derivatives pathway.
Amino acids are used to synthesize proteins, peptides, and other nitrogen-containing substances uniquely required by the organism [22].Amino acids are also degraded into α-keto acids [23], amines [24], and carbon dioxide through deamination, transamination [25], Fig. 7 KEGG Enrichment Analysis.The size of the circle represents the number of genes enriched to the pathway, the larger the circle represents the more number of genes, and the color represents the significance, the deeper the color represents the lesser P value.P < 0.05 indicates significant difference.V: metabolic pathways enriched on 27 and 29 profile, VI: metabolic pathways enriched on 40, 26, 38 and 37 profile, VII: metabolic pathways enriched on 46 and 48 profile Fig. 6 Gene sets with significant trends in STEM analysis.The positions of the dots represents a sample, the lines represents gene changes from the previous sample to the subsequent sample, and the lines of different colors represents different genes.C group was supplement of 0% PKM.L group was supplement of 15% PKM.M group was supplement of 18% PKM.H group was supplement of 21% PKM.P < 0.05 indicates highly significant difference and a combination of deamination or decarboxylation.The α-keto acids can be converted into glucose, lipids, re-synthesized into some non-essential amino acids [26], or oxidized to carbon dioxide and water through the tricarboxylic acid cycle to release energy.PSMC3, PSMC5, PSMD4, PSME1, ACADSB, ECHS1, SDS, OTC, and PAH were the main DEGs in this study.These genes were categorized into three major groups comprising proteasome family genes (PSMC3, PSMC5, PSMD4, PSME1), acyl coactivator genes (ACADSB, ECHS1), and genes that code for enzymes that regulate the metabolism of specific amino acids (SDS, OTC, and PAH).Component of the 26 S proteasome (PSMC) [27] is a multiprotein complex involved in ubiquitin-dependent protein degradation (Fig. 9a).This complex cycle plays a key role in maintaining protein homeostasis by removing misfolded or damaged proteins.Proteasome 26 S Subunit Ubiquitin Receptor (PSMD4) acts as a ubiquitin receptor subunit by interacting with ubiquitin motifs, where it selects ubiquitin conjugates for destruction.Thus, PSMD4 displays a preference for polyubiquitin chains [28].Protein degradation mediated by the proteasome is essential for protein homeostasis and is strongly dependent on proteasome activator subunit (PSME) genes [29].The three families of genes play an important role in the tricarboxylic acid cycle of the amino acid metabolic pathway.The reason for the significant expression of ATP kinase genes may be because the organism fully absorbs protein in the PKM diet.In oxidative metabolic activity organisms, the tricarboxylic acid cycle in the mitochondria requires large amounts of ATP to accomplish the digestion and absorption of nutrients, resulting in the differential expression of genes at the molecular level.
Short/branched chain acyl-CoA dehydrogenase (ACADSB) plays an important role in L-isoleucine metabolism by catalyzing the dehydrogenation of 2-methylbutyryl-CoA [30], one of the components of the L-isoleucine catabolic pathway.The protein encoded by the enoyl-CoA Hydratase-Short Chain 1 (ECHS1) gene functions in the second step of the mitochondrial fatty acid beta-oxidation pathway [31], where it catalyzes the hydration of 2-trans-enoyl-coenzyme A (CoA) intermediates to L-3-hydroxyacyl-CoAs.These two genes ACADSB and ECHS1 are lysosomes that participate in the conversion of α-ketoglutarate to CoA after the production of α-ketoglutarate from amino acids in the first step of amino acid catabolism (Fig. 9c).The products of these genes promote amino acid catabolism.The Serine Dehydratase (SDS) gene encodes one of three enzymes involved in metabolizing serine and glycine.L-serine dehydratase converts L-serine to pyruvate and ammonia.Serine Dehydratase (SDS) can also metabolize threonine to NH4 + and 2-ketobutyrate.Ornithine Transcarbamylase (OTC) encodes a mitochondrial matrix enzyme [32].The protein is involved in the urea cycle, where it converts ammonia into urea for excretion.Phenylalanine Hydroxylase (PHA) encodes a member of the biopterin-dependent aromatic amino acid hydroxylase protein family [33].The phenylalanine hydroxylase enzyme hydroxylates phenylalanine to tyrosine, which regulates phenylalanine catabolism (Fig. 9b).Diseases associated with PHA include Phenylketonuria and Hyperphenylalaninemia.Differential expression of the discussed genes indicated that palm kernel meal diets participate in the amino acid decarboxylation and deamidation processes, significantly impacting the activity of other amino acids.How palm kernel meal regulates the expression of these genes is currently unknown, and this requires further investigation.Understanding the mechanism by which PKM promotes amino acid metabolism in the liver will accelerate its utilization as a feed supplement.

Conclusions
Supplementing 18% PKM in diet significantly enhanced the antioxidation and immune response capacity in Tibetan sheep by enhancing the functioning of hepatocytes.At the molecular level, PKM increased the expression of genes involved in the amino acid metabolism in the liver.This study had one main limitation, it did not investigate the mechanisms by which PKM regulates amino acid metabolism.Further studies are needed to explore the molecular mechanism by which PKM promotes amino acid metabolism.

Fig. 1
Fig. 1 Effect of varying supplement levels of PMK on hepatic phenotypes.C group was supplement of 0% PKM.L group was supplement of 15% PKM.M group was supplement of 18% PKM.H group was supplement of 21% PKM.HE staining, 400x.a: Interlobular veins, b: Interlobular artery, d: Sinus Periphery Gap, e: Hepatic Blood Sinusoids, f: Macrophages

Fig. 3 Fig. 2
Fig. 3 PCA Analysis.C group was supplement of 0% PKM.L group was supplement of 15% PKM.M group was supplement of 18% PKM.H group was supplement of 21% PKM.P < 0.05 indicates highly significant difference

Fig. 4
Fig. 4 Differential Expressed Genes.Vertical bars shows the number of intersections, lines between points represents specific intersecting groups, and horizontal bars represents the amount of raw data for individual groups.C group was supplement of 0% PKM.L group was supplement of 15% PKM.M group was supplement of 18% PKM.H group was supplement of 21% PKM.III: Differential Expression Genes Venn Diagram, the overlap is co-annotated to the gene.IV: Gene expression UpSet Diagram, The black dots represent the samples, the line connecting the dots represents the sample intersection portion, the vertical bars represent the number of intersections, and the horizontal bars represent the sample raw volume

Fig. 5
Fig. 5 Short Time-Series Expression Miner (STEM) Analysis.A box represents a class of gene sets, a colored box represents a gene set with a significant trend, and the same color represents the same expression trend.C group was supplement of 0% PKM.L group was supplement of 15% PKM.M group was supplement of 18% PKM.H group was supplement of 21% PKM.

Table 1
Concentrate supplement and nutrient levels in diet (dry matter basis %)

Table 2
Antioxidant and immune response index Note Data in the same line with the same or no lowercase letters indicate nonsignificant differences (p > 0.05), and data with different lowercase letters indicate significant differences (p < 0.05).* represent to significant difference (P < 0.05)

Table 3
Basic sequencing date statistics for each sample